LCRMP-1 is required for spermatogenesis and stabilises spermatid F-actin organization via the PI3K-Akt pathway

Long-form collapsin response mediator protein-1 (LCRMP-1) belongs to the CRMP family which comprises brain-enriched proteins responsible for axon guidance. However, its role in spermatogenesis remains unclear. Here we find that LCRMP-1 is abundantly expressed in the testis. To characterize its physiological function, we generate LCRMP-1-deficient mice (Lcrmp-1−/−). These mice exhibit aberrant spermiation with apoptotic spermatids, oligospermia, and accumulation of immature testicular cells, contributing to reduced fertility. In the seminiferous epithelial cycle, LCRMP-1 expression pattern varies in a stage-dependent manner. LCRMP-1 is highly expressed in spermatids during spermatogenesis and especially localized to the spermiation machinery during spermiation. Mechanistically, LCRMP-1 deficiency causes disorganized F-actin due to unbalanced signaling of F-actin dynamics through upregulated PI3K-Akt-mTOR signaling. In conclusion, LCRMP-1 maintains spermatogenesis homeostasis by modulating cytoskeleton remodeling for spermatozoa release.


Reviewer #1 (Remarks to the Author):
This study examined the physiological role of LCRMP-1, which showed strongest expression in the testis, by producing knockout mice and studied the phenotype of them closely. The KO mice showed normal development and growth but the sperm production was significantly reduced. This reduction was then revealed caused by abnormal spermiation. As the LCRMP-1 was supposed to take roles in actin dynamics, authors examined the signaling cascade regarding to F-actin organization to show upregulation of some relating molecules.
This study was performed with sophisticated manner resulting clear and solid data, supporting the conclusion of this manuscript.
I have noticed some minor concerns in the manuscript as described below.

Response:
Thank the reviewer's affirmation. We realize that any concerns from the reviewer aim to improve the quality and reliability of our study. We had revised the manuscript according to the reviewer's comments and suggestions point-by-point in below. Thank you for your professional comments. What pathological scores actually represent is also not clear. The lesions in an organ would be completely different from those in other organs. The score 5 = severe/high is added with 76-100%, which is also confusing.

Response:
We really apologize our unclear and inappropriate illustration of Supplementary Table 1. Actually, regarding the pathology report of mice, it was judged by the veterinarian of Animal Center of College of Medicine, National Taiwan University. The evaluation criteria and interpretation were based on the previous study 1 . However, it was our mistake that we did not clarify each number in the report. The grading levels by pathologists may vary greatly. It should be relatively easy to allocate lesions into severity grades relative to the percentage of the tissue/organ affected. Although the nomenclature used for severity grading varies between laboratories, it appears that the most commonly used grading schemes include four or five severity grades to which either descriptive terms (minimal, mild, moderate, etc) and/or numerical levels (grade 1, 2, 3, etc.) were applied. Therefore, we utilized "0", "1", "2", "3", "4", and "5" to represent not present, <1%, 1-25%, 26-50%, 51-75%, and 76-100% of the tissue/organ affected, respectively. In order to disclosure and clarify our evaluation, we fully revised our Supplemental Table 1 with appropriative footnotes and the corresponding description in our manuscript (Line 110-111 and 452-456). Thank you very much.

Line 452-456
The H&E staining and histopathological interpretation of mouse organs was performed by the pathologist from the Taiwan Mouse Clinic and Laboratory Animal Center, College of Medicine, National Taiwan University Medical College. Each organ section was examined at lower-power field and quantified with severity grading scheme (Grading scheme I) based on the previous study 1 .
2. Line 120, Fig. 2b; The tubular diameter was measured to be around 500 µm in this experiment. It is strange however that regular mouse seminiferous tubules are having about 200 µm in diameter. I suspect that authors used incorrect measure or miscalculated.

Response:
We deeply apologized this obvious due to incorrect measurements. In previous measurements, we did not pay attention to variations in shape of seminiferous tubules including round and oval caused by cutting plane of sections. To correct this bias, we referred to the previous study to recount 2 (Fig. 2e).

Line 456-461
The diameter of seminiferous tubule calculation was according to the previous study 2 .
Briefly, only the round or nearly round cross sections of the seminiferous tubules were chosen randomly and measured for each group. The diameter of the seminiferous tubule was measured and averaged across the long and short axes. Totally 180 round tubules from 6 mice each group were obtained for analysis.
3. Line 122 -135; The story line in this part is irregular and a bit confusing. A simple conclusion seems to be "reduced number of spermatozoa in KO mice" in the sample collected from epididymis. Reduced motility is another abnormal data. Other than those, spermatozoa in epididymis were similar to those in control. Such fact can be written in a simple way.

Response:
We really apologized for the confused story line. Be honest, it was also difficult for us to decide the storytelling in our previous submission. Since the reviewer also had such an opinion, we reorganized and rewrote this paragraph in our manuscript to increase ease of understanding and avoid confusing (revised Fig. 2 and Line 119-136).
We can conclude that mice deficient in LCRMP-1 exhibited reduced number of spermatozoa. Please check it. Thank you very much. a The size of testes and epididymides and the ratio of their weights to body weight (n = 7, mean ± SD). b The viability of sperm was analyzed using the eosin-nigrosin staining method. Viability was calculated as the number of white sperm cells divided by the total number of sperm cells (200 sperm cells for each mouse sample; n = 12, mean ± SD). Scale bars, 10 μm. c The concentration of spermatozoa in male mice of different ages (6-8 weeks and 6-7 months). The numbers were counted with a hemocytometer (n = 11 for 6-8 weeks, n = 5 for 6-7 months, mean ± SD, two-tailed Student's t-test, ** p < 0.01, *** p < 0.001). group. The diameters of 30 round tubules from each mouse were measured, and the mean diameters were calculated by averaging the long diameter and short diameter (n = 6, mean ± SD, two-tailed Student's t-test, *** p < 0.001). Scale bars, 100 μm.

Line 119-136
To elucidate the cause of this reduced fertility, we further evaluated macroscopic and microscopic characteristics including quantity and quality of testes and sperm based on previous histopathological analysis. There were no significant differences in the appearance, weight of the testes, sperm viability, and sperm morphology of Lcrmp-1 −/− and Lcrmp-1 +/+ mice (Fig. 2a, b). However, there were substantially fewer spermatozoa in adult Lcrmp-1 −/− , even in mice aged 6-7 months, this substantial reduction was obvious (Fig. 2c). We further tested the function of the spermatozoa. It was notable that the motility of the sperm in Lcrmp-1 −/− was significantly lower (Supplementary Movie 1). By using the OpenCASA software analysis, the sperm from Lcrmp-1 −/− exhibited fewer fast trajectories than Lcrmp-1 +/+ sperm (Fig. 2d). Except for motility, other 4. Line 149, Fig. 3c; The staining of SCP3 seems not specific to spermatocytes, but even the interstitial tissue stained also. I think the antibody is not good enough. At closer look, the DDX4 antibody was not so specific to germ cells either.

Response:
Thank the reviewer's reminding. We also noticed this specificity issue. In order to clarify and address whether antibodies conferred to these confusing results, we totally compared additional clones of SCP3 and DDX4. Also, we further adjusted staining procedures especially the antibody dilution buffer. After confirming of antibody issue, we further recalculated positive cell percentage of SCP3 and DDX4 in Lcrmp-1 +/+ and

Response:
We thank the reviewer for stimulating us to think deeply. The reviewer raised an interesting possibility to interpret our novel finding. We went further to find out whether there is any relevant theory that can support the phenomenon we found. This allows for a comprehensive discussion of the paper.
We agreed the reviewer's opinion that the decrease in late-stage germ cells in Lcrmp-1 - accumulate. According to current data, it is still unclear whether the reduction of tubular diameter in LCRMP-1 -/mice conferred to accumulation of early-stage germ cells.
Taken together, we had added several sentences in Discussion for enriching the content of our study (Line 283-286). Thank you very much.

Line 283-286
Also, it cannot be excluded that the decrease in late-stage germ cells due to enhanced apoptosis created an open space for early-stage germ cells to accumulate. These resulted in the accumulation of DDX4-positive cells near the adluminal compartment.
6. Line 171 -175; Relating to the above comment, the Fig. 4c showed that number of ES was higher in KO mice than control, while RS and PS were almost same in both groups. Doesn't this result contradict with data in Fig. 3, which insisted increased number of early-stage germ cells, including PS, in KO mice?

Response:
We apologize for the confusing in the description of the manuscript and figures.
In Fig. 3, we found DDX4 immature germ cells were significantly increased in seminiferous tubules of Lcrmp-1 -/mice (Fig. 3e). In the mention of heterogenous cell types in seminiferous tubules, to further address the distribution area and impact range of immature germ cells, we utilized BSA density gradient sedimentation for fractionation according to the previous study 3 . Based on the density, from top to bottom layers, four major layers were classified including elongating spermatids fractions (ES), round spermatids fractions (RS), pachytene spermatocytes fractions (PS), and other fractions (Others). The "ES" fraction means that the fraction should normally and mainly contain more mature spermatids.
In Fig. 4c, we had found that the DDX4-postitive immature germ cells were 2.07and 1.08-fold significantly increased in ES and RS but not PS fractions, respectively, of Lcrmp-1 -/mice compared with those of Lcrmp-1 +/+ mice (revised Fig. 4c). Therefore, we suggested that immature germ cells increased in Lcrmp-1 -/possibly due to LCRMP-1 deficiency-mediated accumulation of early-stage germ cells. These were consistent with the conclusion of Fig. 3.
To avoid confusing and misunderstanding, we had revised not only the label of  7. Line 216; "nearer" may be near.

Response:
We highly apologize this low-level error. We had corrected it in our revised manuscript (Line 221-224). Thank you for the reminding.
This should be confirmed by protein expression of LCRMP-1 and CRMP1 in testes from wild-type and knockout animals by immunoblot analysis.

Response:
We thank the reviewer mentioned this issue. Of course, to confirm the selective expression pattern as shown in RNA level by immunoblot analysis is reasonable and ideal. To be honest, in our original submission, we had tried to confirm the protein expression of LCRMP-1 by Western blot. However, there were objective difficulties including: 1. Low abundant LCRMP-1 expression in all organs (except the brain); 2.
The sensitivity of Western blot; 3. The usage of commercial available CRMP-1 antibody which recognized common 497-523 a.a near the C-terminus (Santa Cruz #sc-365345). Therefore, we individually detected mRNA expression of LCRMP-1 and CRMP-1 by specific primers that recognized either exon 1a or exon 1b as an alternative strategy to demonstrate selective expression. In order to meet the reviewer's comment, we tried our hard to reperform Western blot experiments. The result showed that LCRMP-1 was the most abundant in the brain and relative highly expressed in the testis among other organs by LCRMP-1 specific antibody (revised Fig. 1a). The protein expression of LCRMP-1 and CRMP-1 in testis from wild-type and knockout mice was also detected by CRMP-1 antibody. Furthermore, in order to avoid misunderstanding about selective expression, we added the quantification of LCRMP-1 mRNA expression in the brain along with other organs (revised Fig. 1a). The expression of both LCRMP-1 and CRMP-1 in the brain were the highest, up to hundreds of times higher than in other organs. The corresponding description was also added in the manuscript (Line 95-99). We sincerely expected this effort can satisfy the reviewer. We highly appreciated to the reviewer for understanding of the difficulties.

Line 95-99
In addition to the brain, we found that among the other major organs, LCRMP-1 is most abundant in the testes in mRNA and protein levels (Fig. 1a, upper panel). In contrast to the other organs, the expression level of LCRMP-1 is higher than that of CRMP-1 (the mRNA ratio of Lcrmp-1/Crmp-1 and protein expression) in the testes, suggesting that it may play an important role in the male reproductive system (Fig. 1a, lower panel).
2) Genotypes of Fig. 5c panels are missing. Multiple panels of the same stages (4 panels of VII-VIII; 2 panels of IX-X; 2 panels of XI-XII) should be explained.

Response:
Thanks for the reviewer's reminding. We apologized this inappropriate illustration that may lead to misunderstanding. We have added the genotype on Fig. 5c (all eGFP staining in knockout mice). In the mention of panel numbers, actually, we took many photos from each stage. Based on comprehensive observation, LCRMP-1 had the most impact on stage VII-VIII. That was why we showed 2 more panels for VII-VIII. For this comment, we had added more panels for I-III, IV-VI, IX-X and XI-XII stages. The corresponding descriptions were also revised in the revised manuscript (Line 194-205).
Thank the reviewer's carefulness.

Line 194-205
We also analyzed the localization of LCRMP-1 during the cycle of the seminiferous epithelium. Interestingly, there were dynamic changes in the eGFP signal, which represented LCRMP-1 expression in seminiferous tubules of various stages (Fig. 5c).
In late stage VIII, spermiation is completed and the preleptotene spermatocytes start transiting the BTB. LCRMP-1 was expressed in spermatocytes (especially primary and secondary) that were distributed in the adluminal area surrounded by the BTB area (Fig.   5c, stage VII-VIII). In stages IX-XII, the expression of LCRMP-1 gradually moved to the spermatids and residual bodies (Fig. 5c, stage IX-X and stage XI-XII). It was strongly expressed in the residual bodies during stages I-VI (Fig. 5c, stage I-III and stage IV-VI). In early stage VII, LCRMP-1 expression gradually "diffused" from the center of the lumen to the basement membrane site (Fig. 5c, stage VII-VIII). At the same time, it was also highly expressed along the dorsal curvature of the spermatid head ( Fig. 5c, stage VII-VIII, arrowheads).
3) The expression of LCRMP-1 in seminiferous tubules is shown by eGFP immunohistochemistry (IHC) of LCRMP-1 KO in Fig. 3b. This may be sufficient to show stage-specific LCRMP-1 expression, however, eGFP signal cannot represent the subcellular localization of LCRMP-1. Subcellular localization of LCRMP-1 in developing spermatids along with F-actin-staining should be presented with higher magnification.

Response:
We highly appreciated the reviewer raised this important issue about LCRMP-1 subcellular localization. We agree that eGFP staining of original 3b was insufficient to address this issue. We performed LCRMP-1, phalloidin, and DAPI staining in seminiferous tubules followed by confocal microscope analysis with 63x objective lens (revised Fig. 3c and original Fig. 3c and 3d were extended to Fig. 3d and 3e, respectively). In addition, we also analyzed this issue in other stages of developing spermatids. The result was responded to other comments of the reviewer (please see below). In revised Fig. 3c, we found that LCRMP-1 was cytoplasmic localization and partially colocalized with phalloidin. The corresponding description was also added in the revised manuscript (Line 148-154). Thanks for the reviewer's reminding.
Note: We also provided an additional immunofluorescence image by confocal microscope with higher magnification (100x objective lens) in this response letter for the reviewer's reference in below. Please check it.

100x magnified immunofluorescence image
4) It is difficult to image the illustrated F-actin distribution (Fig. 6a right

Response:
Thank the reviewer's reminding. We totally agreed that it was difficult to image the illustrated F-actin distribution only based on the staining of original Fig. 6a left and middle panels. The low power field was not sufficient to fully support our description.
To further provide solid evidences, we had reperformed experiments of triple immunostaining with Sertoli cell marker, SOX9, Phalloidin, and DAPI followed by confocal microscopy analysis under 100x magnification view according to the reviewer's recommendation (revised Fig. 6b). These results further support the illustration of original Fig. 6a right panel. We highly appreciated the reviewer guided us to improve our results. To descript our results more clearly, we reorganized the order of figures. We combined the new triple immunostaining (revised Fig. 6b) with original Fig. 6a left panels (revised Fig. 6a) and original Fig. 6a right panels (revised Fig. 6c) as Fig. 6. The original Fig. 6b and 6c (Western blot analysis) were shifted to the new Fig. 7 (new Fig. 7). This rearrangement aims to separate storytelling for different types of results. The corresponding descriptions were added and changed in our revised manuscript (Line 225-235). Thank the reviewer's understanding.

Line 225-235
To further characterize the organization of F-actin in Lcrmp-1 −/− mice, immunofluorescence triple staining of SOX9 (Sertoli cell), phalloidin (F-actin), and DAPI followed by confocal microscope analysis was performed (Fig. 6b). The results showed that F-actin was well-organized forming a nest-like or fence-like structure between the dorsal of spermatid head and the nuclei of the Sertoli cell that facilitated the removal of the residual bodies from spermatids in Lcrmp-1 +/+ mice in stages VII-VIII while F-actin appeared wrapping around the heads and parts of tails of spermatid in Lcrmp-1 -/- (Fig. 6b, arrowheads). In addition, in stages IX-X, the condensed and bundled F-actin appeared surrounding the tails of elongating spermatids in Lcrmp-1 +/+ mice while the diffuse and disrupt F-actin organizations were observed in Lcrmp-1 -/- (Fig. 6b, arrowheads). These results indicated that LCRMP-1 deficiency caused disorganization of F-actin resulting in aberrant spermiation (Fig. 6c). Fig. 6b and 6c panels should include LCRMP-1 and CRMP1 immunoblots.

Response:
Thank the reviewer's reminding. We apologized this carelessness. We sincerely hoped that we can obtain the understanding of the reviewer that this experiment had objective difficulties due to either the trace amount of LCRMP-1 in testis fractions or high background noise and interference in BSA fractionation. Even so, we still tried our best to perform this experiment to meet the reviewer's requirement. We hoped this can satisfy the reviewer. With the consideration of other comments, we had rearranged the order of figures. Original Fig. 6b and 6c had been changed to Fig. 7a and 7b (revised (n = 3, mean ± SD, two-tailed Student's t-test, * p < 0.05, ** p < 0.01, *** p < 0.001).

Line 237-261
The imbalanced Akt-mTOR-p70S6K/GSK3β axis interferes F-actin dynamics during spermatogenesis in mice deficient in LCRMP-1 To further understand the mechanism by which LCRMP-1 mediates the process of spermiation, we focused on cytoskeletal arrangements in the late stages of spermatogenesis. Based on previous studies, we hypothesized that the F-actin dynamics and organization required for normal spermiation can be stabilized by either ribosomal protein S6 kinase (p70S6K) or LCRMP-1, regulated by protein kinase B (Akt)downstream mammalian target of rapamycin (mTOR) and glycogen synthase kinase-3 beta (GSK3β), respectively 5,6 . Based on a previous finding that Akt is a key molecule regulating the ectoplasmic specialization dynamics in the seminiferous tubules at various stages 7 , we first tested the signaling involved in Akt-mediated F-actin dynamics (Fig. 7a). The expression of LCRMP-1 as control, compared with Lcrmp-1 +/+ mice, phosphorylated Akt (p-Akt) and upstream phosphorylated phosphoinositide 3kinase (p-PI3K) signaling were significantly upregulated in the testes of Lcrmp-1 −/− mice. The expression of downstream molecules, p-mTOR and p-GSK3β, was also enhanced as a result of Akt activation. Furthermore, we observed increased levels of cleaved caspase 3 in the testes of Lcrmp-1 −/− mice, suggesting that germ-cell apoptosis may be the consequence of spermiation failure. Considering all the effects on heterogenous cell populations in the testis that are caused by LCRMP-1 deficiency, we separated testicular cells into high-and low-LCRMP-1-expressing fractions (HEF and LEF, respectively), based on LCRMP-1 and eGFP expression, for further validation (Fig. 7b). We found that p-Akt, p-mTOR, p-GSK3β, and p-p70S6K were upregulated in the LEF of Lcrmp-1 −/− mice, while cleaved caspase 3 was significantly upregulated in their HEF. Combined, these results suggest that LCRMP-1 deficiency causes F-actin disorganization during late spermatogenesis, followed by spermiation failure and germcell apoptosis. LCRMP-1 deficiency may cause imbalanced regulation by two Aktdownstream signaling pathways, enhancing the mTOR-p70S6K signal and resulting in the F-actin disorganization phenotype in stages VII-X ( Supplementary Fig. 6).
6) Several scenarios for the molecular machinery of compensatory upregulation of Aktsignaling in the absence of LCRMP-1 may be proposed in the discussion.

Response:
Thank the reviewer's reminding. We will add several scenarios to mention the molecular machinery which may be involved in compensatory upregulation of Aktsignaling in the absence of LCRMP-1.
In previous studies, CRMPs proteins were known as cargo adaptors connecting kinesin with cargo proteins such as Sra-1/WAVE-1 complexes or interacting with factors promoting polymerization regulating axon formation or cell adhesion. [8][9][10][11] . It is possible that LCRMP-1 may serve as either a cargo adaptor transporting the Sra- We had added these possible scenarios in the discussion to rich the content of manuscript (Line 317-337). According to these inputs, we had revised and reorganize whole Discussion to avoid redundance. Please check it. Thank you very much.

Line317-337
Previous studies showed CRMPs served as either a cargo adaptor transporting the Sra- 7) L237, eGFR should be eGFP

Response:
We highly apologize this error. We had corrected it in our revised manuscript (Line 252-255). Thank you for the reminding.

Line 252-255
Considering all the effects on heterogenous cell populations in the testis that are caused by LCRMP-1 deficiency, we separated testicular cells into high-and low-LCRMP-1expressing fractions (HEF and LEF, respectively), based on eGFP expression, for further validation (Fig. 7b).
I appreciate that authors responded positively to my comments and the manuscript was improved from my point of view.
There are yet several points that I would like to comment on, as shown below.
Line 31-33: "In conclusion, LCRMP-1 maintains homeostasis of spermatogenesis by consummating spermiation, and the Lcrmp-1−/− mouse model provides a potential strategy for investigating male reproductive system." This last sentence in the abstract seems a bit out of focus and not appropriate as summarizing the study. The latter half of the sentence, in particular, is questionable how the Lcrmp-1−/− mouse model can provide a strategy for investigating such a huge research area of male reproductive system. Line 170: The reference 23 is about the in vitro spermatogenesis using an organ culture method. In this reference paper I did not find DDX4 or VASA. I suspect that the reference is incorrect.
Line 185: "In mice, the complete process of spermatogenesis takes about 8.6 days, and different stages of spermatogenesis occur in each segment of the seminiferous tubules." The complete process of spermatogenesis in mice takes 35 days, not 8.6 days. The 8.6 days are a period taken by a single cycle of the seminiferous epithelium takes place.
Line 283-286; Although authors have taken my comments in the revised manuscript of this part, "an open space" hypothesis may be inappropriate here. I'm sorry but now I think this sentence may be better to be omitted, if authors agree.
Reviewer #2 (Remarks to the Author): Comments for revised manuscript of "LCRMP-1 consummates spermiation in male reproduction by stabilizing F-actin organization via PI3K-Akt pathway balancing".
Very little is known about the molecular mechanism of spermatogenesis. Chang J-H et al. revealed that long-form collapsing response mediator protein-1 (LCRMP-1) is involved in the spermatogenesis based on their findings including abundant expression of LCRMP-1 in testis and reduced fertility of LCRMP1 knockout (KO) mice. They found that LCRMP-1 is localized in spermiation machinery at the late stages of spermatogenesis. Using the LCRMP-1 KO mice, they showed that the absence of LCRMP-1 brings disorganized F-actin assembly in the late spermatogenesis, spermiation failure and germ-cell apoptosis, and the upregulation of Akt-mTOR signaling. In this revised manuscript, they discussed possible molecular mechanisms connecting LCRMP-1 and spermiation through Akt-mTOR pathway. The authors have earnestly addressed all of the comments raised by reviewers #1 and #2. I am satisfied with their revisions and find these results very interesting. This study becomes as a significant contribution.

Response summary
The following letter contains full point-by-point responses to both reviewers (5 from the reviewer #1 and 1 from the reviewer #2). We briefly summarized responses in below with a hyperlink to each detail description within the letter.
For the convenience of reviewers to read, we additionally provided a clear manuscript file with line number labeled as the supplemental information. The line number in the response letter was based on this supplementary file.

Response:
Thank the reviewer's comment. We agree with the reviewer that "the Lcrmp-1−/− mouse model provides a potential strategy for investigating male reproductive system" may be suspected of a little bit overinterpretation. Also, this statement may be out of focus and not appropriate as summarizing our finding. Therefore, we had modified the last sentence as "In conclusion, LCRMP-1 maintains homeostasis of spermatogenesis by consummating spermiation by modulating cytoskeleton remodeling for spermatozoa release" (Line 30-32). Thank you.

Line 30-32
In conclusion, LCRMP-1 maintains homeostasis of spermatogenesis by consummating spermiation by modulating cytoskeleton remodeling for spermatozoa release. Some readers may feel in the same way.

Response:
Thank the reviewer's comments. We agreed with the reviewer that many readers may be confused to our description and raise the same challenge. And we apologize for the confusion. It should be noticed that BSA density isolation allowed cells to be separated based on specific gravity.
Therefore, in normal circumstances, the mature and immature germ cells should be localized to corresponding fractions (layers). How could the DDX4-positive immature germ cells in Lcrmp-1 -/mice have been "trapped" in ES and RS fractions especially the ES fraction? We think there are three possibilities. One is the number of DDX4-positive immature germ cells, which were spermatogonia, spermatocytes, and early spermatids 1 , increased in Lcrmp-1 -/mice so that some cells overflowed into ES and RS fractions. Another is that the differentiation was interfered by increased immature germ cells due to LCRMP-1 deficiency so that each stage cells stacking was out of original order with alterations of gravity (density). The other is that, as mentioned by the reviewer, the expression of DDX4 might be maintained in late-stage spermatids due to LCRMP-1 deficiency. However, without further evidences, we prefer the first possibility based on: 1. The morphology of DAPI staining indicated DDX4-positive cells were similar to immature germ cells; 2. On the basis of LCRMP-1 characteristics, it may not affect the DDX4 expression directly.
Taken together, with the reviewer clarification, we agreed that the present description may lead to some confusions and misunderstandings. We had corrected our sentences in the revised manuscript (Line 279-282). Thank you very much.

Line 279-282
In particular, the presence of more DDX4-positive immature germ cells in the fractions where LCRMP-1 should have been highly expressed suggested that the accumulation of these cells overflowed into these fractions.

3.
Line 170: The reference 23 is about the in vitro spermatogenesis using an organ culture method. In this reference paper I did not find DDX4 or VASA. I suspect that the reference is incorrect.

Response:
We apologized our unclear description. The reference 23 was cited due to the guidance for us to differentiate the different stages of germ cells by different nuclear morphology. Our apologies, we did not cite the reference related to DDX4-expression pattern for identification of different germ cells at the end of this sentence. We added additional references and please check it out (Line 167-170). Thank you so much!

4.
Line 185: "In mice, the complete process of spermatogenesis takes about 8.6 days, and different stages of spermatogenesis occur in each segment of the seminiferous tubules." The complete process of spermatogenesis in mice takes 35 days, not 8.6 days. The 8.6 days are a period taken by a single cycle of the seminiferous epithelium takes place.

Response:
We thank the reviewer's correction. We are sorry for our carelessness and mistake. We have corrected the sentence (Line 185-189). Thanks for the reviewer's kindly reminding.

Line 185-189
In mice, the complete process of spermatogenesis takes about 35 days, which is four times longer than the period of a single seminiferous epithelial cycle from stage I to stage XII (8.6 days), and different stages of spermatogenesis occur in each segment of the seminiferous tubules. This pattern is called the "cycle of the seminiferous epithelium", which is divided into 12 stages (I-XII) 4-6 .

5.
Line 283-286; Although authors have taken my comments in the revised manuscript of this part, "an open space" hypothesis may be inappropriate here. I'm sorry but now I think this sentence may be better to be omitted, if authors agree.

Response:
Thank the reviewer's understanding. Actually, we appreciated that the reviewer raised this hypothesis previously. It was interesting for us and promoted us to have in-depth thinking. Since we had no further evidence or data to support this hypothesis, we agreed with the reviewer that we omitted this discussion at this moment (Line 284-285). We highly appreciated the reviewer's inputs.

Line 284-285
It is possible that numbers of meiotic and early-stage germ cells, which are DDX4 positive, accumulated due to abnormal spermiation in Lcrmp-1 -/mice. Also, it cannot be excluded that the decrease in late-stage germ cells due to enhanced apoptosis created an open space for early-stage germ cells to accumulate. These resulted in the accumulation of DDX4-positive cells near the adluminal compartment.

Reviewer #2 (Remarks to the Author):
Comments for revised manuscript of "LCRMP-1 consummates spermiation in male reproduction by stabilizing F-actin organization via PI3K-Akt pathway balancing".
Very little is known about the molecular mechanism of spermatogenesis.

Response:
We thank the reviewer's careful reminding. We apologized this mistake and had revised it (Revised Fig. 5).